Mammalian spermatozoa contain choline acetyltransferase (ChA), acetylcholine (ACh), acetylcholinesterase (AChE) and a cholinergic receptor (ChR). They also contain phosphatidylethanolamine (PE) and phosphatidycholine (PC). PE may be converted to PC by two phosphatidyl-N-methyltransferases. Choline may be liberated from PC through a sequence of enzymatic steps. This liberated choline may be converted to ACh, which acts on ChR to regulate sperm motility. Thus, ACh synthesis may be linked to phospholipid metabolism. The primary objective of this investigation is to demonstrate the interrelationships between phospholipid methylation and ACh function in spermatozoa. These studies give the following results: (1) Spermatozoa contained two phospholipid methyltransferases which converted PE to PC by stepwise methylation. Zn ions was an inhibitor of phospholipid methylation and may serve as a modulator of these reactions. 2-Benzoylethyltrimethyl-ammonium (BETA) was a selective inhibitor of ChA and also sperm motility. (2) Spermatozoa contain methionine enkephalin which may modulate the action of ACh on sperm motility. Further work is in progress on the following: (a) Effects of ACh agonists and antagonists on phospholipid methylation, membrane fluidity and sperm motility; (b) Sources of choline for ACh synthesis in spermatozoa: choline uptake systems, base exchange mechanisms involving PC and choline; (c) Effects of tertiary ChA inhibitors in sperm motility in vitro and in vivo; and (d) Effects of antifertility agents (cyproterone acetate, alpha-chlorhydrin and trimethylphosphate) on the levels of ACh, ChA and acetate thiokinase, ATP citrate lyase, and phosphatidyl-N-methyltransferases in epididymal spermatozoa in the rat. These investigations may be useful to (1) explain the role of ACh and PC in the regulation of sperm motility; (2) understand certain types of infertility in men who ejaculate immotile sperm; and (3) develop antifertility agents.